The pistil is the reproductive organ of the angiosperm flower which harbours the female gametophyte (Esau K., Plant Anatomy, John Wiley, New York (1965)). In spite of its critical role in plant reproduction, relatively few genes have been identified which are predominantly expressed in pistils and most of the characterized genes function primarily within the transmitting tissue (Gasser C. S., Robinson-Beers K., Plant Cell 5:1231-1239 (1993)) or are associated with self-incompatibility (Nasrallah J. B., Nasrallah M. E., Plant Cell 5:1325-1335 (1993); Newbigin E., Anderson M. A., Clarke A. E., Plant Cell 5:1315-1324 (1993); Trick M., Heizmann P., Int. Rev. Cytol 140:485-524 (1992)).
In order to isolate additional genes which are important to pistil function and development, a Brassica napus flower cDNA library constructed in .lambda.gt10 was differentially screened as described previously (Robert L. S., Allard S., Franklin T. M., Trick M., Mol. Gen. Genet 242:209-216 (1994)). One cDNA clone Pis 63 represented a gene highly expressed in the pistil and was investigated further (Robert L. S., Allard S., Gerster J. L., Cass L., Simmonds J., et al., Plant Mol. Biol. 26:1217-1222 (1994)). Northern blot analyses showed that Pis 63 transcripts were found primarily in the stigmatic region and that they could be detected in stigmas throughout pistil development starting with low levels in 1-2 mm flower buds. Pis 63 transcripts were still present one week post anthesis, but were absent in the stigma dissected from a 4 cm silique.
In situ hybridization performed on median longitudinal sections of 3 mm developing flower buds showed that Pis 63 expression was limited to the stigmatic cells of the pistil. A .sup.35 S-labelled Pis 63 antisense ribo-probe hybridized strongly to the papillar cells of the stigma, whereas no differential hybridization was observed with the sense RNA probe. No hybridization was detected in the style or the ovary.
A limited number of genes have been shown to function primarily in the stigma and they include proteinase inhibitors from Nicotiana alata (Atkinson A. H., Heath R. L., Simpson R. J., Clarke A. E., Anderson M. A., Plant Cell 5:203-213 (1993)), chitinases from Petunia hybrida (Leung, D. W. M., Phytochemistry 31:1899-1900 (1992)) and genes involved in the sporophytic self-incompatibility system of Brassica (Goring D. R., Glavin T. L., Schafer U., Rothstein S. J., Plant Cell 5:531-539 (1993); Nasrallah J. B., Nasrallah M. E., Plant Cell 5:1325-1335 (1993); Robert L. S., Allard S., Franklin T. M., Trick M., Mol. Gen. Genet 242:209-216 (1994); Trick M., Heizmann P., Int. Rev. Cytol 140:485-524 (1992); and PCT application WO 94/25613.
The Brassica pistil is considered to have a dry stigma with the external surface of its papillar cells being covered by a protein pellicle (Mattson O., Knox R. B., Heslop-Harrison J., Heslop-Harrison Y., Nature, 247:298-300, 1974). Since the Pis 63 gene product may be secreted, it could form a constituent of the papillar protein pellicle and hence be involved in encouraging the germination of compatible pollen or discouraging pathogen infection. The relevance of the similarity between the Pis 63 protein and a protein expressed in cotton fibre remains to be elucidated. This cotton protein encoded by pCEK6 may play either a structural role, or a role in the biosynthesis or degradation of polysacharides within cotton fibre (John M. E., Crow L. J., Proc. Natl. Acad. Sci USA 89:5679-5773 (1992). It is possible that both these proteins serve similar functions as structural or defence proteins.
Accordingly, it is thus desirable to isolate the promoter region from a genomic clone corresponding to said cDNA clone, wherein said promoter region could be used to direct high levels of transcription to the stigma.